Polyamine Titers Research Articles

The effects of exogenous Ca 2+ on endogenous polyamine levels and drought-resistant traits of spring wheat grown under arid conditions

This study investigates the effects of exogenous Ca 2+ on physiological and phenotypic characteristics of spring wheat ( Triticum aestivum L.) in an arid region of China. These traits, which are closely linked to drought tolerance in plants, include alteration in endogenous levels of polyamines (PAs), root-to-stem ratios and relative water content (RWC). Exogenous Ca 2+ was applied to the seeds by soaking in a 0.5% CaCl 2 solution before sowing and also by spraying the leaves with the same solution. It was determined that the foliar RWC increased at all stages of development following the Ca 2+ application. The levels of endogenous PAs in the leaves were altered: the putrescine (Put) content decreased whereas the spermidine (Spd) and spermine (Spm) content increased. The ratio of Put to PAs titers was reduced. At the same time, phenological events of wheat were also changed in response to the application of Ca 2+. In particular, reproductive maturity was significantly delayed. Moreover, the application of Ca 2+ increased both the root-to-shoot ratios of wheat at the early stages of growth and the final grain yield. The results obtained from this study show that the exogenous Ca 2+ could not only enhance the tolerance of spring wheat to drought but also delay its senescence. Finally, the role of Ca 2+ in the signal regulation of endogenous PA metabolism is discussed. We suggest that exogenous Ca 2+ may have a role in modifying endogenous PAs levels under drought stress leading to increased drought tolerance.

Expression of an antisense Datura stramonium S-adenosylmethionine decarboxylase cDNA in tobacco: changes in enzyme activity, putrescine-spermidine ratio, rhizogenic potential, and response to methyl jasmonate

S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermidine and spermine synthesis through specific synthases which use putrescine, spermidine and decarboxylated S-adenosylmethionine as substrates. In order to better understand the regulation of polyamine (PA), namely spermidine and spermine, biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco (Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S promoter, by means of Agrobacterium tumefaciens and leaf disc transformation. The effect of the genetic manipulation on PA metabolism, ethylene production and plant morphology was analysed in primary transformants (R0), and in the transgenic progeny (second generation, R1) of self-fertilised primary transformants, relative to empty vector-transformed (pBin19) and wild-type (WT) controls. All were maintained in vitro by micropropagation. Primary transformants, which were confirmed by Southern and northern analyses, efficiently transcribed the antisense SAMDC gene, but SAMDC activity and PA titres did not change. By contrast, in most transgenic R1 shoots, SAMDC activity was remarkably lower than in controls, and the putrescine-to-spermidine ratio was altered, mainly due to increased putrescine, even though putrescine oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls. Despite the reduction in SAMDC activity, the production of ethylene, which shares with PAs the common precursor SAM, was not influenced by the foreign gene. Some plants were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown second generation transgenic plants, at the vegetative stage, SAMDC activity was scarcely reduced, and PA titres did not change. Finally, the rhizogenic potential of in vitro-cultured leaf explants excised from antisense plants was significantly diminished as compared with WT ones, and the response to methyl jasmonate, a stress-mimicking compound, in terms of PA conjugation, was higher and differentially affected in transgenic leaf discs relative to WT ones. The effects of SAMDC manipulation are discussed in relation to plant generation, culture conditions and response to stress.

Effect of salt and osmotic stress on changes in polyamine content and arginine decarboxylase activity in Lupinus luteus seedlings

The effects of NaCl (260 mM) and sorbitol (360 mM) isoosmotic stresses on polyamine titers in lupin (Lupinus luteus L. var. Ventus) in relation to organ-specific responses were investigated. Analysis showed that during the first few hours (4 h) of salt and osmotic stress higher amounts of putrescine (Put) and spermidine (Spd) were accumulated in the roots and leaves of lupin seedlings. After exposing the plants to a longer duration (24 h) of exposure to NaCl, the level of free Put decreased in roots and cotyledons by about 48% and 54%, respectively, and increased in hypocotyls and leaves by about 27% and 73%, respectively. The Level of free Spd also decreased in roots by about 50%, in contrast to the increase of Spd observed in hypocotyls and leaves by about 50% and 70%, respectively. The effect of non-ionic stress on the level of Put and Spd in studied organs of lupin was similar to that of NaCl. Free spermine was at an undetectable level in examined organs. However, in the roots of lupin growing for 24 h in the presence of NaCl and/or sorbitol, the activity of arginine decarboxylase (ADC) (EC 4.1.1.19) increased by about 66% and 80%, respectively. ADC activity in leaves was similar to that observed in the control. Additionally, in the roots and leaves of lupin growing under the stress condition (NaCl or sorbitol), a higher level of polyamines (PAs) bound to microsomal membranes was observed. It is probable that PAs bound to microsomal membranes prevent stress-induced damage. We conclude that both stresses induce biosynthesis of Put and other PAs in the roots, as well as Put accumulation in the leaves, and this may indicate translocation of Put from the roots to the shoot. The possible role of PAs in adaptive mechanisms to stress is discussed.

Changes in polyamine pattern are involved in floral initiation and development in Polianthes tuberosa

In the day-neutral plant Polianthes tuberosa (cv. Double) putrescine and spermine in corms at the early floral initiation stage decreased by 26 and 36%, respectively, compared with that in the vegetative stage. In contrast, a sharp increase in spermidine and cadaverine titers in corms was recorded at the early floral initiation stage. However, cadaverine in corms disappeared at the flower development stage. Polyamines in the roots were generally lower than those in the leaves and corms. In no case was the change in endogenous polyamine titers in the roots and leaves associated with floral initiation and flower development in P. tuberosa. Exogenous application of spermidine at 5, 25 or 150 microg per plant at the vegetative stage did not affect flower primordium counts. However, addition of a spermidine synthase inhibitor, cyclohexylamine, at 150 or 250 microg per plant (each dose was applied two times in total at an interval of 4 days) significantly reduced flower primordium counts, indicating that spermidine is involved in floral initiation and floral development in P. tuberosa. In P. tuberosa corms at the vegetative stage arginine decarboxylase activity rises and decreases at the early floral initiation stage. In contrast, ornithine decarboxylase activity reaches the highest level at the early floral initiation stage and declines significantly at the vegetative stage. Results indicate that an increase in spermidine and a transient increase in cadaverine titers in the corms seem characteristic of early floral initiation in P. tuberosa. It is also suggested that a significant reduction in putrescine and spermine in the corms is involved in the early floral initiation in P. tuberosa.

Peach ( Prunus persicaL.) fruit growth and ripening: transcript levels and activity of polyamine biosynthetic enzymes in the mesocarp

Transcript levels and activities of the polyamine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19), ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), as well as free polyamine titres, were analysed throughout the four growth stages S1-S4 leading up to ripening in the mesocarp from peach fruit (Prunus persica L. Batsch cv. Redhaven) grown under field conditions. SAMDC mRNA, which was northern analysed by using a PCR-generated homologous SAMDC probe, and ADC mRNA levels appeared quite stable during fruit development, while ODC transcript accumulation showed a discontinuous trend. The pattern of transcript levels during growth did not correlate with that of the relative enzyme activity, which instead correlated well with free polyamine levels. Both exhibited maximum levels in S1 and a smaller peak in S3. The behaviour of the polyamine biosynthetic machinery is discussed in relation to the different cell growth rates occurring during fruit development.

Alteration in polyamine titres during Agrobacterium-mediated transformation of indica rice with ornithine decarboxylase gene affects plant regeneration potential

We report the changes in polyamine titres during the Agrobacterium-mediated transformation of rice calli and their influence on plant regeneration capacity following the introduction of putrescine biosynthesis gene ornithine decarboxylase ( odc) as well as β-glucuronidase ( gus) reporter gene. Free polyamine titres increased during the Agrobacterium infection and a general increase in the polyamine titres was seen during early stages of selection. The changes in polyamine levels reflective of the transgene expression emerged late during selection and continued during the regeneration from the calli. An increase in the putrescine titres was observed during regeneration and was found to be helpful for regeneration. These results suggest that polyamine metabolism responds to transformation as well as transgene expression and provided a tool for improving the regeneration from transformed rice calli by modulating polyamine metabolism.

Role of polyamines in the ontogeny of plants and their biotechnological applications

Recent developments in the metabolism and function of polyamines in plants is presented. Polyamines appear to be involved in a wide range of plant processes, however their exact role is not completely understood. In this review, the metabolic pathways involved in polyamine biosynthesis and degradation are explained, along with the transport and conjugation of these compounds. The studies involved in the understanding of function(s) of polyamines using metabolic inhibitors, as well as genetic and molecular approaches are described. Polyamine metabolism and profound changes in polyamine titres in response to infection by pathogens has been presented. Its role in adaptation of plants to stress is also presented. Molecular understanding of polyamines and their modulation in transgenics is also discussed. Further line of work in the understanding of the role of polyamines has also been focussed.

Polyamines as biomarkers for plant regeneration capacity: improvement of regeneration by modulation of polyamine metabolism in different genotypes of indica rice

The importance of cellular polyamine (PA) levels and the ratio of putrescine (Put) to spermidine (Spd) for plant regeneration ability via somatic embryogenesis in several commercially grown indica rice varieties is reported here. The genotypes namely NDR-624, IR-20, IR-36, BJ-1 (having Put:Spd ratio∼2.3) showed superior plant regeneration while KL, PB-1 and TN-1 (having Put:Spd ratio∼3.8) showed moderate plant regeneration ability. The genotypes namely HS, Bindli, DV-85, ACB-72, IR-64 and IR-72 (having Put:Spd ratio∼5.0) showed poor plant regeneration ability. In contrast KH-7 (Put:Spd ratio∼10.0) showed no response at all. Favorable modification of cellular PA titers and their Put:Spd ratio by the addition of exogenous PAs (Put, Spd) or their biosynthesis inhibitor, difluoromethylarginine (DFMA) led to the induction/promotion of plant regeneration in poorly responding genotypes. These results showed a close relationship between cellular PA levels and their Put:Spd ratio with in vitro morphogenetic capacity in indica rice and suggest that the cellular PAs and Put:Spd ratios are important determinants (biomarkers) of plant regeneration ability in indica rice, and the improvement/induction of plant regeneration in morphogenetically poor and recalcitrant species could be achieved by modulating PA metabolism.

Putrescine and Silver Nitrate Influences Shoot Multiplication, In Vitro Flowering and Endogenous Titers of Polyamines in Cichorium intybus L. cv. Lucknow Local.

The influence of putrescine (Put) and AgNO(3) on shoot multiplication, in vitro flowering and endogenous titers of polyamines in Cichorium intybus L. cv. Lucknow local was investigated. Exogenous administration of Put at a concentration of 40 mM resulted in maximum tissue response in terms of shoot numbers (34.6 +/- 2.61) and shoot lengths (7.6 +/- 0.57 cm) on MS media supplemented with 2-iP (2.0 mg L(-1)) and GA(3) (0.5 mg L(-1)) as observed on the 35(th) day. Exogenous application of 40 µM AgNO(3) resulted in maximum shoot number (36.8 +/- 2.63) and shoot lengths (7.9 +/- 0.76 cm) on day 35 on the same media. Endogenous titers of conjugated spermidine decreased sharply from day 7-21, whereas endogenous conjugated spermine levels peaked on day 28 (1265 +/- 94.9 nmoles g(-1) FW), after treatment with 40 mM Put. Whereas, AgNO(3) (40 µM) fed samples resulted in higher titers of endogenous conjugated spermine (1405 +/- 105.6 nmoles g(-1) FW, 3.62 fold over control) on day 14. All other treatments showed decreasing endogenous levels during the whole culture period. Both Put (40 mM) and AgNO(3) (40 µM) resulted in floral initiation and floral development on day 28 and 14 (3.76 +/- 0.16, 4.2 +/- 0.21 flowers per shoot apices), respectively. To investigate the role of Put (40 mM) and AgNO(3) (40 µM) on morphogenetic response and endogenous conjugated polyamine titers in shoots of chicory, polyamine inhibitors (DFMA and DFMO) were used. The morphogenetic response and the endogenous conjugated pool of polyamines were diminished in DFMA and DFMO treatments, but could be restored by addition of Put (40 mM) and AgNO(3) (40 µM). Under exogenous Put feeding, ethylene production was reduced in shoot cultures of chicory. This study shows for the first time the influence of polyamines on multiple shoot initiation from axillary buds of C. intybus L. cv. Lucknow local and also indicates the promotive effect of Put and AgNO(3) on autoregulation of polyamine biosynthesis, thereby regulating in vitro flowering, the endogenous pool of polyamines and shoot multiplication.

A role for spermidine in the bolting and flowering of Arabidopsis

Several lines of experimental evidence indicate a close connection between polyamines (PAs) and reproductive development in Arabidopsis thaliana. (l) Measurement of the titers of endogenous spermidine (SPD) and putrescine (PUT), extracted from various organs of two ecotypes and a genetic line of Arabidopsis, revealed that flowers had the highest titers of both PAs, with SPD predominating. (2) In aseptic cultures of whole plants of the ecotype Columbia, the application of appropriate enzyme inhibitors lowered SPD titer while almost completely preventing bolting and flowering. When the plants were removed to an inhibitor‐free medium, bolting and flowering resumed. (3) SPD added to the medium of aseptically cultured plants of Columbia growing under short‐day (SD) conditions, where flowering is naturally delayed, increased the SPD titer and augmented the rate and extent of flowering. Under long‐day (LD) conditions, where flowering is already rapid and abundant, it did not promote flowering any further. (4) Enzyme inhibitors of SPD synthesis given shortly before the transition from SD conditions to LD conditions prevented flowering. (5) In a delayed‐flowering mutant (CS 3123), the addition of SPD significantly accelerated flowering.

Organogenesis fromSolanum melongena L. (eggplant) cotyledon expiants is associated with hormone-modulated enhancement of polyamine biosynthesis and conjugation

Eggplant (Solanum melongena L. cv. Violetta lunga 2) cotyledon expiants grown on hormone-free medium (controls) or on medium containing either naphthaleneacetic acid alone (root forming) or in combination with zeatin riboside (shoot forming) showed minor differences in free polyamine titres during culture. In contrast, conjugated polyamines (particularly those in the trichloroacetic acid-soluble fraction) accumulated only in hormonetreated explants, but not in controls. The extent and the temporal changes in soluble-conjugate levels differed between root-forming and shoot-forming expiants; in the former, accumulation began earlier (within 1 day of culture) and reached the highest levels. In both organogenic programmes, maximum conjugate accumulation occurred just before and during organ emergence. Adventitious roots and shoots were formed along the cut surfaces. The regions closest to these (“borders”) displayed a significantly higher ratio of conjugated to free spermidine and/or putrescine than the nonorganogenic regions (“centres”) of the explant. Ornithine decarboxylase activity was higher than arginine decarboxylase activity both in control and hormone-treated explants. However, both activities increased markedly on day 2 of culture in the presence of hormones. Thereafter ornithine decarboxylase activity remained high in shoot-forming explants, but not in root-forming ones. Putrescine oxidising activity was also enhanced by exogenously supplied hormones starting from day 4 of culture. This activity remained high up to day 12 in the presence of auxin plus cytokinin, whereas it peaked on day 6 in auxin-treated explants. Spermidine oxidising activity was the only enzyme activity which was consistently higher in controls than in hormone-treated tissue. Differences between the two organogenic programmes with respect to temporal changes in polyamine content, and putrescine biosynthetic and oxidative activities are discussed in relation to the timing of organ formation. The latter was monitored both histologically and macroscopically.

Enhancement of Growth and Coumarin Production in Hairy Root Cultures of Witloof Chicory (Cichorium intybus L.cv.Lucknow local) under the Influence of Fungal Elicitors.

Studies were conducted to determine the possible production of coumarins in hairy root cultures of Cichorium intybus L.cv. Lucknow local under the influence of microbial agents. Pythium aphanidermatum and Phytopthora parasitica var. nicotiana were cultured and their mycelial and medium filtrate were used for the elicitation of coumarin production. The media filtrate of P. parasitica at 1.0% v/v added to the MS basal medium led to the maximum growth of hairy roots of C. intybus, which was 1.57-fold higher than the untreated control on the 28th day of culture, along with maximum esculin and esculetin yields which were 4.06- and 3.71-fold higher than the control on the 28th day. The yields of esculin and esculetin in hairy root cultures of chicory strongly correlated with growth. To check the effect of these fungal elicitors on endogenous polyamine metabolism, titers of total endogenous polyamines were analyzed. It was confirmed that the media filtrate of P. parasitica at 1.0% v/v concentration resulted in maximum accumulation of total endogenous polyamines, wherein endogenous spermine titers were found to be maximum as compared to endogenous spermidine and putrescine titers on the 28th day. Total endogenous spermine in the case of 1.0% MF v/v P. parasitica was 1.3-fold higher than that of the control on the 28th day of culture. Maximum growth index with greater length of primary root (17.61+/-0.18 cm) and greater number of secondary and tertiary roots was recorded for the medium filtrate of P. parasitica at 1.0% v/v concentration. This study also provided an insight into the morphological changes in terms of branching patterns, occurring in roots under the influence of these fungal elicitors.

Enhancement of growth and coumarin production in hairy root cultures of witloof chicory ( Cichorium intybus L.cv.Lucknow local) under the influence of fungal elicitors

Studies were conducted to determine the possible production of coumarins in hairy root cultures of Cichorium intybus L.cv.Lucknow local under the influence of microbial agents. Pythium aphanidermatum and Phytopthora parasitica var. nicotiana were cultured and their mycelial and medium filtrate were used for the elicitation of coumarin production. The media filtrate of P. parasitica at 1.0% v/v added to the MS basal medium led to the maximum growth of hairy roots of C. intybus, which was 1.57-fold higher than the untreated control on the 28th day of culture, along with maximum esculin and esculetin yields which were 4.06- and 3.71-fold higher than the control on the 28th day. The yields of esculin and esculetin in hairy root cultures of chicory strongly correlated with growth. To check the effect of these fungal elicitors on endogenous polyamine metabolism, titers of total endogenous polyamines were analyzed. It was confirmed that the media filtrate of P. parasitica at 1.0% v/v concentration resulted in maximum accumulation of total endogenous polyamines, wherein endogenous spermine titers were found to be maximum as compared to endogenous spermidine and putrescine titers on the 28th day. Total endogenous spermine in the case of 1.0% MF v/v P. parasitica was 1.3-fold higher than that of the control on the 28th day of culture. Maximum growth index with greater length of primary root (17.61±0.18 cm) and greater number of secondary and tertiary roots was recorded for the medium filtrate of P. parasitica at 1.0% v/v concentration. This study also provided an insight into the morphological changes in terms of branching patterns, occurring in roots under the influence of these fungal elicitors.

Regulation of ethylene and polyamine synthesis by elevated carbon dioxide in cherimoya fruit stored at ripening and chilling temperatures

In this study we focused on the effect of high CO2 level (20%) on ethylene and polyamine biosynthesis in cherimoya (Annona cherimola Mill.) fruits stored at ripening (20˚C) and chilling (6˚C) temperatures. At ripening temperature, CO2 inhibited ethylene production, but 1-aminocyclopropane-1- carboxylate (ACC) oxidase activity was similar to that in ripe control fruits. CO2 treatment led to a decline in putrescine (Put) and a major accumulation of spermidine (Spd) and spermine (Spm) without any effect on arginine decarboxylase (ADC) activity. These results confirm the preferential transformation of Put to Spd and Spm in CO2 -treated fruits. At chilling temperature, the increase in ACC oxidase activity was inhibited and the Vmax of ADC increased. A combination of chilling temperature storage and high CO2 level led to suppression of basal ethylene production while ACC oxidase activity remained unchanged. In addition, fruits held at these conditions had higher polyamine titres than the untreated control. We propose that, in CO2 -treated fruits, the absence of autocatalytic or basal ethylene production, depending on the temperature, may be due to deviation of the S-adenosylmethionine (SAM) pool towards polyamine synthesis, primarily Spd and Spm.

Ripening Affects High-Temperature-Induced Polyamines and Their Changes during Cold Storage of Hybrid Fortune Mandarins

Polyamine levels were measured in the flavedo of three ripening stages of Fortune mandarins exposed to high-temperature conditioning before storage at a chilling (2 °C) and a nonchilling temperature (12 °C). Increasing periods of conditioning at 37 °C delayed the development of chilling injury (CI) and produced an increase in the content of the polyamines in the following proportion: spermine > spermidine > putrescine. Storage at 2 °C of conditioned fruits reduced the polyamine titer to levels similar to those of nonconditioned fruits. Changes in polyamines during storage of conditioned and nonconditioned fruits were dependent on the ripening stage of the fruit. In general, the levels of polyamines in nonconditioned fruits stored at 2 and 12 °C were similar. No change in spermine was induced in the most mature fruits at 2 °C or in response to high-temperature conditioning despite conditioning-reduced CI. With this exception, the data indicate that conditioning fruits at 37 °C elevated the content of poly...

Effect of high CO2 level on the titres of -aminobutyric acid, total polyamines and some pathogenesis-related proteins in cherimoya fruit stored at low temperature

In this study the effect of treatment with 20% CO2 plus 20% O2 for 3 d on γ-aminobutyric acid (GABA) was analysed and total polyamine titres and the production of some pathogenesis-related proteins (PR-proteins) in cherimoya (Annona cherimola Mill.) fruit stored at low temperature. In immunoassays with anti-PR-Q and PR-2 protein sera, high CO2 levels were found to provoke the co-ordinated accumulation of a chitinase-like protein and 1,3-β-glucanase. Chitinase activity was higher in treated than in untreated fruit. At the end of CO2 treatment, total polyamine and γ-aminobutyric acid content and uptake of O2 were observed to be higher in treated compared to untreated fruit, but the accumulation of these compounds decreased when the fruit was transferred to air. Since this treatment effectively retains the fruit quality (Escribano et al., 1997), high CO2 levels may have a direct effect on the activation of the above-mentioned specific responses that enables cherimoya fruit to overcome chilling temperature. The relationship between the activation of the defence system and the capacity to regulate cytoplasmic pH in CO2-treated fruits was also addressed.

The effect of methionine, ethylene and polyamine catabolic intermediates on polyamine accumulation in detached soybean leaves

In the present study we determined the effects of methionine, intermediates of polyamine catabolic pathways and inhibitors of either ethylene biosynthetic or polyamine catabolic pathways on polyamine accumulation in soybean leaves. Inhibitors to SAM decarboxylase and spermidine synthase, methylglyloxal‐bis‐(guanylhy‐drazone) and cyclohexylamine, respectively, suggest that methionine may provide aminopropyl groups for the synthesis of polyamine via S‐adenosylmethionine (SAM). Results from experiments that utilized a combination of compounds which altered either ethylene or polyamine biosynthesis, namely, aminoethoxyvinyl glycine, CoSO4, 2,5‐norbornadiene, and CuSO4, suggest the two pathways compete for a common precursor. However, exogenous addition of ethylene (via ethephon treatments) had little or no effect on polyamine biosynthesis. Likewise, polyamine treatments had little or no effect on ethylene biosynthesis. These data suggest that there are few or no inhibitory effects from the end products of one pathway on the synthesis of the other. Data from leaves treated with metabolic intermediates in the catabolic pathway of polyamines and inhibitors of enzymes in the catabolic pathway, i.e. aminoguanidine, hydroxyethyldrazine and gabaculine, suggest that the observed increases in polyamine titers were not due to decreased catabolism of the polyamines. One catabolic intermediate, γ‐aminobutyric acid (GABA), elevated putrescine, spermidine and spermine by 12‐, 1.4‐, and 2‐fold, respectively, Ethylene levels decreased (25%) in GABA‐treated leaves. This small decrease in ethylene could not account for such large increase in putrescine titers. Further analysis demonstrated that the GABA‐mediated polyamine accumulation was inhibited by difluoromethylarginine, an inhibitor of arginine decarboxylase, but not by difluoromethylornithine, an inhibitor of ornithine decarboxylase. These data suggest that GABA directly or indirectly affects the biosynthesis of polyamines via arginine decarboxylase.

Polyamine-bound trypsin inhibitors in sweet potato (Ipomoea batatas [L.] Lam cv. Tainong 57) storage roots, sprouted roots and sprouts

Trypsin inhibitors (TIs) were purified from storage roots, sprouted roots and sprouts of sweet potato variety Tainong 57 (T57) by ammonium sulfate precipitation and Sephadex G-75 chromatography. Active fractions were further purified by affinity chromatography on trypsin-Sepharose 4B. Activity staining of TIs on a 15% SDS-PAGE gel revealed TI bands (73, 38 and 22 kDa) in storage roots, sprouted roots and sprouts. TIs, purified by the affinity column, were hydrolyzed by mixing with an equal volume of 12 N HCl at 110°C for 16 h. The hydrolysates were benzoylated with benzoyl chloride in alkaline condition, and the polyamines (PAs) were identified by HPLC using 64% methanol as an eluent. Cad, Spd and Spm were found in all TI hydrolysates with different amounts in storage roots, sprouted roots and sprouts. TIs purified from the sprouts had higher PA titers, which were expressed as nmol/μg protein, than those from sprouted roots or storage roots. The possible physiological roles of PA-bound TIs are discussed.

Effects of cadmium on the host-pathogen system. V. Effect of exogenous dicyclohexylamine on potato tubers ( Solanum tuberosum L.), cadmium and Phytophthora infestans relations

Summary Treatment of potato ( Solanum tuberosum L.) tuber slices with dicyclohexylamine (DCHA) led to a reduction in some of endogenous polyamine (PA) titers and peroxidase (PO) activity in and to a decrease in the electrolyte leakage (EL) from the tissues. Simultaneously, these changes were associated with the alteration of interactions between this tissue and cadmium, and Phytophthora infestans . Two cultivars, Bintje and Bzura, respectively susceptible and resistant to cadmium and pathogen, were examined. Pretreatment of tuber slices with DCHA, before inoculation, resulted in limitation of infection in the susceptible tissues. The possible involvement of PA in the transduction of the cadmium and pathogen signals is discussed.

Spatial distribution of free and conjugated polyamines in leaves ofSolanum melongenaL. associated with differential morphogenetic capacity: efficient somatic embryogenesis with putrescine

In eggplant (Solanum melongena L., cv. Pusa Purple Long), explants from different regions of the leaf showed significant differences for embryogenic potential. Discs from the apical region of leaves yielded more somatic embryos than those from the basal region. Apical discs showed consistently higher polya-mine titres than the basal discs. Putrescine treatment promoted somatic embryogenesis and at 0.5 mM it caused a remarkable increase (c. 6-fold) in a number of somatic embryos, accompanied by an increased putrescine titre. On the other hand, spermidine and spermine had no stimulatory effect on embryogenesis; rather they were inhibitory at higher concentrations. All tested inhibitors of polyamine biosynthesis such as difluoromethylarginine, difluoromethylomithine, methylglyoxal bis (guanylhydrazone) and bis (cyclohex-ylammonium) sulphate significantly inhibited somatic embryogenesis. Difluoromethylarginine blocked somatic embryogenesis by lowering endogenous polyamine contents (particularly putrescine) and such inhibitory effects were totally restored by exogenous putrescine (0.5 mM), concomitant with the revival of endogenous PA concentrations. These results demonstrate (i) a positive correlation between the spatial distribution of free and conjugated polyamines and the embryogenic capacity of an explant and (ii) putrescine caused the promotion of somatic embryogenesis, suggesting the intricate regulatory role of polyamines, specifically putrescine, in somatic embryogenesis in eggplant.